Send Orders of Reprints at [email protected] Current Medicinal Chemistry, 2012, 19, 4451-4461 4451 As Therapeutics with Enhanced Bioactivity

D. Goodwin1, P. Simerska1 and I. Toth*,1,2,

1The University of Queensland, School of Chemistry and Molecular Biosciences; 2School of Pharmacy, St. Lucia 4072, Queensland, Australia Abstract: The development of techniques for efficient production renewed interest in peptides as therapeutics. Numerous modi- fications for improving stability, transport and affinity profiles now exist. Several new adjuvant and carrier systems have also been developed, enhancing the immunogenicity of peptides thus allowing their development as vaccines. This review describes the established and experimental approaches for manufacturing peptide drugs and highlights the techniques currently used for improving their drug like properties. Keywords: Peptide, drug delivery, vaccine, manufacture, bioavailability, peptide therapeutic, immunogenicity, peptide drug, peptide synthe- sis, clinical trials.

INTRODUCTION side-chain reactivity, degree of modification, incorporation of un- natural components, in addition to the required purity, solubility, Natural and synthetic peptides have shown promise as pharma- stability and scale. There are two strategies for peptide production - ceutics with the potential to treat a wide variety of diseases. This chemical synthesis and biological manufacturing. potential is often overshadowed by the inability of the peptides to reach their targets in an active form in vivo. The delivery of active Chemical peptides is challenging due to inadequate absorption through the Chemical synthesis has been used for the production of peptides mucosa and rapid breakdown by proteolytic enzymes. Peptides are in both research and industry and led to the development of the usually selective and efficacious, therefore need only be present in majority of peptide drugs [2]. These chemical-based strategies were low concentrations to act on their targets. The of pep- usually favored due to their unique ability to incorporate any num- tides is superior to other small molecules due to the limited possi- ber of non-natural components. Two chemical methodologies- bility for accumulation and result in relatively non-toxic amino solution-phase (SPS), and solid-phase peptide synthesis (SPPS) [4] acid, peptide metabolites. These properties contribute towards the - are the predominant methods of chemical synthesis, but many overall low toxicity of peptides, with a limited risk of adverse inter- other approaches, including hybrid synthesis and chemoselective actions. ligation, have also been studied. The most important classes of peptides that have been investi- Solution-Phase Peptide Synthesis gated include , gonadotropin-releasing , , the enkephalins, the , other hormone analogs, enzyme First described by du Vigneud in 1953, the SPS method was inhibitors and vaccines. The majority of peptides were targeted used for the production of most commercial peptide therapeutics towards treatment of cancers and metabolic disorders, with a large [5]. The method was based on the coupling of peptide fragments or number targeting G- coupled receptors. In 2010, there were a single in solution Fig. (1). The main advantage of SPS over 50 peptide drugs approved for marketing, with annual global was economic because of the use of relatively inexpensive building sales nearing/surpassing US$ 1 billion for the following peptide blocks and reagents. Each intermediate could be analyzed and puri- drugs: ciclosporin (e.g. Neoral®, Novartis), goserelin acetate (Zo- fied, leading to products of higher purity and easier final purifica- ladex®, AstraZeneca), glatiramer acetate (Copaxone®, Teva Phar- tions. The main limitation of SPS was the need to modify each maceuticals), leuprolide acetate (e.g. Lupron®, Abbott Laborato- intermediate reaction step to synthesize a required peptide thus, the ries), and octreotide acetate (Sandostatin®, Novartis). Additionally, development of each synthetic scheme was long, laborious, and in 2010, more than 100 peptide drug candidates were reported to be relatively expensive. Peptide pharmaceuticals from as few as 3 in clinical development [1]. amino acids (e.g. thyrotropin-releasing hormone) to peptides of 32 residues (e.g. calcitonin) have been synthesized for clinical use This review describes novel approaches in peptide synthesis using SPS [2]. and the use of peptides as therapeutics. Solid-Phase Peptide Synthesis PEPTIDE THERAPEUTICS Ten years after the development of SPS, Merrifield introduced SPPS [6]. This method was based on the coupling of protected Initially, inefficient and expensive manufacturing processes amino acids to an insoluble support Fig. (1). The simplicity of this hampered the development of peptide-based therapeutics. Neverthe- process was compatible with automated synthesis for the large scale less, the production of the peptide drug enfuvirtide (Fuzeon®, production of peptides. SPPS proved to be faster and less laborious Roche, a human immunodeficiency virus (HIV) membrane fusion than SPS. Two forms of SPPS methods were developed based on inhibitor), demonstrated that preparing a 36-amino acid peptide on the protection of the -amino group of an amino acid, the tert- a ton-scale annually, could be cost effective [2]. butyloxycarbonyl (Boc) group was deprotected by acid (e.g. The establishment of industrial-scale peptide manufacturers trifluoroacetic acid) [7], while 9-fluorenylmethyloxycarbonyl meant that the chemical industry had to supply increased demands, (Fmoc) group was removed by base (e.g. piperidine) [8]. which caused a decrease in prices, increased availability of amino The most important limitation to the use of SPPS on an indus- acids and consequently, market growth [3]. trial scale was the requirement for excess starting material [2]. The methods used for the preparation of peptide therapeutics However, several peptide therapeutics such as (Prialt®, have depended on a number of important criteria: sequence length, Elan), exanatide (Byetta®, /Eli Lilly Co.), (Symlin®, Amylin) and degarelix (Firmagon®, Ferring) were syn- *Address correspondence to this author at the School of Chemistry and Molecular thesized by SPPS and approved by the U.S. Food and Drug Biosciences, The University of Queensland, St Lucia, Queensland, Australia; 4072, Administration (FDA) [9, 10]. Since 1992, microwave irradiation Tel: +61 7 33469892; Fax: +61 7 33654273; E-mail: [email protected] has been used extensively to accelerate SPPS by providing a con-

1875-533X/12 $58.00+.00 © 2012 Bentham Science Publishers 4452 Current Medicinal Chemistry, 2012 Vol. 19, No. 26 Goodwin et al. venient rapid heating method [11]. SPPS was used to make Grami- Pseudo- (Pro) modified amino acid residues were cidin A (peptide antibiotic) and CSF114(Glc) (a im- designed to minimize aggregation and racemization when synthe- munological probe) with a good yield and high purity [12]. sizing difficult peptide sequences [18]. Pseudo- (Pro) are Hybrid Synthesis - or theonine-derived oxazolidines or -derived thio- zolidines with proline-like ring structures Fig. (2). A fragment con- Due to the time limitations and adverse side-reactions present in densation of the complex RNase1-39 glycopeptide made with Pro traditional SPS and SPPS protocols, synthesis of some peptide fragments was carried out successfully using SPPS without racemi- sequences has often proven difficult. Hybrid synthesis, which com- zation [19]. The use of (PEG)-based resins, bines SPS and SPPS, merged the benefits of both protocols Fig. (1) particularly ChemMatrix® (CM), showed benefits over traditional [4]. The production of enfurvirtide, established using SPPS, was polystyrene-based resins [20, 21]. CM was very stabile with a high changed to a hybrid strategy for manufacture in the scale of metric loading capacity and has been used in both aqueous solutions and tons annually from three or more fragments [2]. during the synthesis of difficult hydrophobic peptides [21]. The use Chemoselective Ligation of Pro in combination with CM was successful in the synthesis of a 68-amino acid chemokine, which was unsuccessful using a poly- Another option for the elongation of peptide chains is through styrene resin due to aggregation [22]. Kessam et al. suggested that a chemoselective ligation techniques, which offer the ability to cou- newly developed resin incorporating PEG polymerized onto a ple unprotected peptide fragments Fig. (1). Native poly(vinyl alcohol) core may be even more valuable, economically was described as one example of chemoselective ligation, which, and environmentally, after demonstrating its promise against other through a thiol exchange and spontaneous rearrangement, ulti- resins in synthesis of a section of acyl carrier protein [23]. mately formed natural peptide bonds between peptide fragments [13]. The limitation of this strategy was the requirement of an N- terminal cysteine and C-terminal thioester. A modified method used NH O auxillary thiols in place of the cysteine residues, which were HN removed at the site of ligation and allowed the formation of a non- R4 N cysteine linkage [14, 15]. R3 O Improving Chemical Synthesis R 2 X H Due to ecological concerns about the impact of chemical syn- R1 thesis, there has been pressure to move towards more environmen- tally friendly methods, or ‘green’ chemistries. The elimination of Fig. (2). General structure of a pseudo-proline (Pro) residue. traditional organic solvents, such as dimethylformamide and dichloromethane, can save considerable toxic waste. Hojo et al. Biological Peptide Synthesis developed a new, water soluble N-protecting group, 2-[phenyl (methyl)sulfonio]ethoxycarbonyl tetrafluoroborate, which was used Biological methods of peptide production countered some of to successfully synthesize Leu- and Met-enkephalin peptides in the shortfalls of traditional chemical synthesis (e.g. sequence length aqueous solution on a TentaGel resin [16]. The same group reported and timeframe concerns). Enzymatic synthesis and native isolation the ability of Boc-protected amino acids to be nanodispersed in were widely used for the assembly of shorter active peptide se- water and used in SPS. These nanodispersed Boc-amino acids cou- quences [24]. Fermentation was used for production of short pep- pled with 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmor- tides, such as cyclosporine [25]. In vivo and in vitro recombinant pholinium chloride and N-methylmorpholine produced the Leu- systems utilized generated fragments of DNA or plasmid DNA to enkephalin pentapeptide in 89% yield [17]. ultimately translate the protein or large peptide of interest. These methods are limited by difficulties in purifying the peptide of

Fig. (1). Principals of chemical syntheses. Each method involved a cycle of deprotection and coupling to form a chain of amino acids. PG: protecting group; AA: amino acid; X: (activated) functional group; R: solid support resin; SPPS: solid-phase peptide synthesis; SPS: solution-phase peptide synthesis; HS: hy- brid synthesis; CL: chemoselective ligation. Peptides As Therapeutics With Enhanced Bioactivity Current Medicinal Chemistry, 2012 Vol. 19, No. 26 4453 interest from the other peptides and from the expression like peptide 1, recombinantly expressed in yeast with a 16-carbon system, and the inability to incorporate a large range of unnatural lipid chain chemically attached to Lys26 via a spacer amino acids. [41, 42]. Televancin (Vibativ®, Theravance/Astellas Pharma) is a Recombinant Synthesis second example of peptide semi-synthesis. Used to overcome resis- tance and to improve ADME properties, televancin is made by the Recombinant synthesis involves the expression of the peptide lipidation of vancomycin (a glycopeptide antibiotic synthesized within a specialized system from an artificial gene. In vivo, this is using fermentation techniques) [43]. In another example of compli- commonly prepared in microorganisms like Eschericia coli (E. coli) cated semi-synthesis, the aforementioned Pro RNase1-39 glycopep- [26]. In some cases, in vivo production took several weeks but the tide fragment condensation product was coupled to a recombi- significant benefit was the ease of upscaling to high-volume fer- nantly-expressed RNase40-124 peptide fragment using native chemi- mentation. In these expression systems, the peptides were usually cal ligation, producing the full protein [19]. fused to carrier domains (e.g. protein) that facilitate their targeting into the periplasm or inclusion bodies. This method allowed for a simpler, rapid purification (e.g. low-speed centrifugation followed by nickel chelation chromatography) of the expressed peptide from the proteins and peptides already existing within the organism. A number of peptide therapeutics prepared by in vivo methods are currently available on the market. For instance, ecallantide (Kalbi- tor®, Dyax Corp.) and desirudin (Iprivask®, Canyon Pharmaceuti- cals), which are 65 and 60 amino acid residue peptides expressed in yeast, and teriparatide (Forteo®, Eli Lilly Co.), 34 residues expressed in E. coli [27-29]. The assembly of a polymeric, tandem- repeat cDNA sequence coding for peptide units separated by stable or cleavable linkers has been used for expressing smaller peptides [30]. A more recent advance in recombinant expression technology was the use of in vitro (cell-free) systems, in which, fragments of DNA were transcribed and translated in a one-pot reaction Fig. (3) [31]. The advantage of these systems is that peptides and proteins can be generated in a short time (minutes to hours). Autonomous cells were used traditionally but a number of alternative organisms such as Leishmania tarentolae (a protozoan parasite of lizards) Fig. (3). Cell-free peptide expression system. Low molecular weight were developed [32] and have been shown to be cheaper than pre- (MW) precursors fed through the dialysis membrane to the molecular ma- vious systems [33]. chinery derived from the cell lysate. The template DNA was added inside The major limitation of biological peptide synthesis is the the membrane with the lysate and translated and expressed in a one-pot inability to incorporate unnatural amino acids. However, this can be reaction. overcome using a range of in vitro expression techniques. Extended anticodon tRNAs (pre-aminoacylated with unnatural amino acids) PEPTIDE VACCINES substituted natural tRNAs to selectively include these amino acids in a number of sequences [34]. One example was the introduction Compared with traditional attenuated vaccines, modern subunit of genetically programmed cross-linking by the presentation of two peptide vaccines have increased safety, higher specificity, greater unnatural residues. Neumann et al. incorporated alkyne- and azide- purity and are more cost-effective to produce. Subunit peptide vac- containing amino acids to facilitate a rapid cycloaddition, to form a cines only include the minimal antigenic components required for triazole cross-linkage for enhanced proteolytic and structural stabi- vaccine efficacy [44]. These peptide-based vaccines are easily lity [35]. modified, non-infectious and highly stabile against enzymatic deg- radation when altered and/or incorporated into carrier [45]. Unlike Other some other formulations, peptide-based subunit vaccines exist as An alternative to using expression vectors is the production of one entity inclusive of all components essential for a successful peptides as hydrolysates from food proteins. This technique has vaccine, incorporating adjuvant, carrier and antigen [46, 47]. This resulted in the production of a range of bioactive peptides. Peptides eliminated the need to co-administer other compounds (e.g. adju- were derived from bovine milk proteins that had angiotensin con- vant) that could be associated with adverse effects in the host. verting enzyme inhibitory, immunomodulatory, antithrombotic and The inherent instability of peptides in peptide-based vaccines hypocholesterolemic activities [36-38]. Enzymes were also applied has the potential to induce non-specific immune responses or may to add functional modifications to peptide precursors (e.g. glycosy- fail to stimulate immunity. Natural peptide epitopes have been sub- lation [39], amidation [40]). Although biological methods have stituted with peptidomimetics and peptide-peptidomimetic hybrids shown considerable promise, these approaches are still generally to enhance specificity and immunogenicity since the early 1990s. expensive and are not necessarily viable for production in industrial The use of peptidomimetics for designing antigens is comprehen- scales. sively reviewed by Croft and Purcell [48]. Combined Chemical and Biological Synthesis: Peptide Semi- Lipopeptide Vaccines synthesis The immunogenicity of peptide vaccines can be improved by Semi-synthesis was developed as the combination of biological the use of lipopeptides. The N-terminal moiety of a lipoprotein and chemical peptide synthesis methods. Peptide sequences pro- found in E. coli, tripalmitoyl-S-glyceryl cysteine or Pam3Cys, was duced by recombinant methods were further altered chemically elucidated in 1983 [49]. The conjugation of bacterial lipid con- and/or enzymatically. One of these examples is , structs, such as Pam3Cys, to synthetic peptides induced high anti- approved by the FDA in 2010 for the glycemic control of type two body titers [50, 51]. The mechanism of action remains unclear, diabetes mellitus in adults. Liraglutide is an analogue of - 4454 Current Medicinal Chemistry, 2012 Vol. 19, No. 26 Goodwin et al. though it was suggested that they assisted in the anchoring, inter- specific antibody titers after intranasal administration. These anti- nalization and presentation of the attached antigens [52]. This was bodies were able to opsonize Group A Streptococci [67]. High anti- due to the action of cell-surface receptors, such as the Toll-like body titers were achieved following subcutaneous administration in receptors, which recognized structurally conserved pathogen- mice [68]. Promisingly, nanoparticle vaccines were shown to pro- associated molecular patterns [45]. vide some degree of protective immunity after mucosal vaccination Another strategy to improve the immunogenicity of peptides [69]. was the incorporation of multiple copies of the peptide in an anti- genic peptide system with a polylysine core [47]. The addition of MODIFICATIONS TO ENHANCE BIOLOGICAL ACTIV- lipids to this system led to the development of the lipid-polylysine ITY OF PEPTIDE THERAPEUTICS core-peptide, LCP Fig. (4). The lipidic tail of the LCP system elic- ited Toll-like receptor activity, which was observed in vitro by its Although peptides have the potential to act as therapeutic drugs, action on the receptor-transfected human embryonic cells their physico-chemical properties often limit their progression from [53]. It also induced in vitro maturation of splenic dendritic cells. research lead to use in the clinic. Fortunately, many physico- Of particular interest, the LCP constructs exhibited significant im- chemical properties can be altered to improve drug delivery and munogenic effects without the need for any additional adjuvant [54, enhance biological activity of therapeutic peptides (e.g. stability, 55]. LCPs were shown to have an additional benefit of storage in transportation and distribution, affinity and targeting, controlled freeze-dried form with resistance to temperature and peptidases, release, and immunogenicity). A particular challenge is the devel- due to qualities of lipidation and the multiple antigenic peptide opment of peptide therapeutics suitable for oral delivery. The gas- system [45]. The versatility of the LCP system has been tested trointestinal tract (GI) impedes delivery of peptide drugs. Along using antigens from Chlamydia trachomatis (C. trachomatis) [56], with cellular peptidases from mucosal cells, the brush-border mem- human papillomavirus [57], and Streptococcus pyogenes [58-62]. In branes of epithelial cells form a major enzymatic blockade [70]. particular, the incorporation of a monomeric peptide derived from Exopeptidases, aminopeptidases and carboxypeptidases in the GI, C. trachomatis incorporated into the LCP system conferred an break down sequences from the N- and C-termini, whereas increase of immunogenicity by up to 3200-fold [56]. When admin- endopeptidases recognized cleavage sites within amino acid istered subcutaneously in mice, these vaccine candidates elicited sequences. The combination of these types of enzymes facilitates high levels of IgG antibodies. Intranasal administration was also rapid and efficient degradation and is a significant barrier to the shown to be successful with this system [63]. administration of peptide therapeutics via the GI route. Acid-labile peptide bonds, such as that of an aspartyl-proline bond [71], con- Peptide tribute to a poor oral bioavailability. Chemical modifications Fig. (5) at positions known to be susceptible to enzymatic cleavage were Lys O reported to increase in vivo stability of peptides-based therapeutics Peptide dramatically. Substitutions with D-amino acids and cyclization Lys NH CH C NH 2 enhanced peptide stability profiles. Other approaches included the Peptide Lys use of other unnatural residues, modification of peptide bonds, ter- mini protection/modifications and conjugation to carrier constructs, Peptide such as biocompatible [72]. Amino acid Peptide backbone n substitutions modifications Antigen Carrier Adjuvant R7 R3 O H H R8 N N Fig. (4). Lipid-core peptide (LCP) system. R4 N-terminal NH R1 The LCP contains all elements of a successful vaccine system: antigen (pep- modification/ C-terminal O R5 O R2 tide), carrier (polylysine) and adjuvant (lipidic tail). conjugation modification/ Side-chain R6 conjugation modification -Lipopeptide Vaccines

In addition to the use of lipopeptides, can also Cyclization improve the immunogenicity of peptide adjuvants. The conjugation of carbohydrate moieties to ovalbumin (OVA, derived from egg Fig. (5). Examples of chemical peptide modifications. R2, R3, R5, R7 = white protein) enhanced antigen presentation [64], while coating Amino acid side chains, proteinogenic or otherwise. Possible modifications: liposome-protamine-DNA nanoparticles with mannan enhanced the R1, R8 = lipids, sugars, acetyls, polymers. R4 = reduced, substituted pepti- immunogenicity of a human papilloma virus-derived peptide epi- domimetic bonds. R6 = methylation, acetylation, hydroxylation. R7 = D- tope [65]. Carbohydrates have also been modified to act as vaccine conformation, tetra-substituted amino acids. carriers, providing variable orientations for the attachment of pep- Solubility, lipophilicity, molecular weight and ionization con- tide antigens. Changing the structure of the carbohydrate carrier tribute most to biodistribution of therapeutics. However, peptides could lead to optimization of the antigen conformation, thus with strong overall positive charges interact with the phosphate enhancing recognition by the immune system. Carbohydrate-based heads of lipids, thus are better able to cross biological membranes LCPs showed promising results; eliciting high IgG antibody titers [73]. Most tested peptides are cleared from circulation within after subcutaneous administration in mice [57, 61]. minutes and oral delivery is rarely possible. Rapid hepatic and renal clearance, aggregation and poor absorption due to hydrophilicity Nanovaccines are the major challenges observed in peptide transport. Organic and inorganic polymers, helical peptides, -sheet form- Peptides were protected from exopeptideases by modification at ing peptides and carbon nanotubes were applied in the design of their N- and C-termini. Examples of modifications were the use of successful peptide-based nanoparticle vaccines. The use of these N-pyroglutamate, C-amidation, acetylation [74], (PEG) systems was recently reviewed by our group [66]. A Group A [75] and conjugation of carbohydrate moieties [76-78]. Glycosyla- Streptococcus peptide epitope was attached to a self-adjuvanting tion at both N- and C-termini conferred improved stability to pep- polyacrylate dendrimer nanovaccine, which produced high epitope- tides [76-78]. Peptides As Therapeutics With Enhanced Bioactivity Current Medicinal Chemistry, 2012 Vol. 19, No. 26 4455

Route of Administration a stable linkage, or a labile one, creating a pro-drug [93, 94]. This use of a pro-drug strategy enhanced the pharmacological character- The route of administration affects the pharmacokinetics of istics of the peptide without decreasing its activity or selectivity peptide drug candidates. Parenteral, oral, mucosal and transdermal [95]. The use of lipidation to form a pro-drug is a useful strategy to routes have been explored, with the parental route being most improve the uptake of peptide therapeutics. However, lipidation common. However, a number of commercial liposome-based tech- may impair peptide delivery by causing poor water solubility. nologies have evidenced the viability of peptide drugs for transder- mal and pulmonary delivery [79, 80]. N-terminal conjugation with lipidic amino acids increased the half-life of LHRH by approximately 30-fold in Caco-2 cell Several non-parenteral routes for peptide delivery bypass the homogenates [92]. The lipidic moiety was cleaved first, releasing gastrointestinal tract, BBB or first-pass metabolism. Formula- the active parent peptide, acting in a pro-drug manner. This specific tion strategies are important in these delivery routes for enhanced cleavage mechanism was also observed in plasma and kidney stability in vivo [81]. Excipients, such as salts, cyclodextrins, preparations [92, 96]. Due to association of the lipid-modified anti- chelating agents and protease inhibitors altered the natural envi- diabetic liraglutide with plasma proteins, plasma half-life of the ronment, slowing down the peptide degradation. peptide increased from 1.2 to 13 hours after subcutaneous admini- The skin is an excellent barrier against large, polar, hydrophilic stration [41]. The peptide drug tesamorelin was approved by the compounds such as peptides. Nevertheless, transdermal delivery FDA in 2010, and used for the treatment of visceral fat accumula- has an advantage over the parental route, enabling control of drug tion in HIV-infected patients with lipodystrophy. Modification of concentration in a non-invasive manner. The modification of lipo- tesamorelin with a hexenoyl moiety enhanced resistance to dipepti- philicity, addition of solvents and a number of physical methods, dyl peptidase 4, which deactivated the parent peptide: growth hor- (ultrasound, iontophoresis and electroporation), have all increased mone-releasing hormone [97]. peptide permeability [82]. Patches containing microprojections Lipidation of hydrophilic compounds has been used extensively have also shown their effectiveness. A successful example of pep- to improve absorption by passive diffusion across epithelial barri- tide vaccine transdermal delivery was the use of dry-coated micro- ers, notably in the gastrointestinal tract and the central nervous projection patches, which required only 1/30 of the dose needed for system [76, 98]. Lipid moieties coupled to peptides conferred the intramuscular administration [83]. These technologies facilitated characteristics essential for intestinal delivery [99]. In studies using the passage of peptides into the epidermis, where it was absorbed a more stable enkephalin analogue, [D-Ala2, D-Leu5]-enkephalin, it into systemic circulation via microcapillaries. A liposome-based was observed that an increased lipid moiety chain length enhanced transdermal system for the delivery of interferon -2b recently pro- its permeability as long as the solubility was not affected [100]. gressed through to phase II/III clinical trials. However, Fredholt et al. observed a decrease in permeability with The large, absorptive surface of the alveoli made pulmonary de- increased lipophilicity in ester pro-drugs [101]. Kellam et al. stud- livery another possible delivery route [84]. Permeation enhancers ied the influence of the carbon chain length on the activity of [D- (e.g. salicylates, fatty acids, bile salts and surfactants) were used to Ala2, D-Leu5]-enkephalin and found that the increased chain length facilitate greater absorption. The potential for long-term negative led to a decreased activity in mouse vas deferens and guinea pig effects, such as irritation and lung damage, and the cost-benefit models [102]. implications of the recently withdrawn inhalable insulin peptide drug Exubera® (Pfizer) made this method less favorable [85]. Due to its vascularized subepithelium, the intranasal route pro- Liposaccharide-based delivery systems were designed to vided a large surface area with high permeability and rapid absorp- improve bioactive peptides [76, 98] and were shown to enhance tion to the systemic circulation. The mucus gel layer and mucocil- their bioavailability [77, 98, 103, 104]. , a peptide with iary apparatus have proven to be considerable barriers to intranasal poor pharmacokinetics, was modified with lipids and with or with- delivery. Absorption enhancers have been used with some success out a monosaccharide moiety. The liposaccharide-modified soma- for overcoming these barriers, however many have been shown to tostatin increased the permeability of the parent peptide when tested damage the sensitive nasal mucosa [86]. Intranasal delivery of insu- in vitro across a Caco-2 cell monolayer [105] without eliminating lin has been thoroughly investigated and a number of nanoparticle the activity of the lead compound. Another example was C-terminal systems have recently shown promise in animal models [87, 88]. In conjugation of a lipid and a monosaccharide attachment on the one example, vasoactive intestinal peptide was shown to reach the N-terminus of a , which increased permeability 60-fold brain when delivered intranasally, whereas intravenous delivery [76]. was unsuccessful, indicating a fraction passed through a direct transport pathway [89]. A number of agonists based on natural pep- Glycosylation was used to target receptors that naturally recog- tides, such as calcitonin (e.g. Miacalcin®, Novartis), desmopressin nize specific carbohydrates, such as lectins. Glycosylated peptides (e.g. Desmospray®, Ferring), -releasing hor- were conveniently targeted to particular tissues or organs, or even mone (LHRH) (e.g. Kryptocur®, Sanofi-Aventis), and specific cells for distinct actions. Arg- was used as a (e.g. Syntocinon®, Novartis), has been approved for intranasal model peptide to explore the possibility of renal targeting. The car- delivery [90, 91]. bohydrate-modified arg-vasopressin peptide bound selectively to microsomal fractions of the liver and kidney [106]. In another Oral delivery is the preferred delivery route due to the ease of study, OVA was used to demonstrate the efficacy of glycosylation administration and greater patient compliance. GI delivery is ham- for the targeting of dentritic cells, increasing antigen presentation pered by concentrated enzymatic activity, the need to pass across by 50-fold for CD4+ T cells and 10-fold for CD8+ in vitro [64]. the intestinal musical epithelia, and the range of pH (1.2-8.0) Nevertheless, liposaccharide modification does not guarantee ideal encountered along the GI tract. Oral delivery of insulin using poly- ADME properties in peptides. mers and emulsions have reached clinical trials [81] and a number of formulations of ciclosporin (e.g. Sandimmune®/Neoral®, Unnatural Amino Acids Novartis) and desmopressin (e.g. DDAVP®, Ferring) have been successfully carried through to the clinic too. The physico-chemical properties of peptides can be improved Lipidation by the substitution of natural amino acids with unnatural ones: D- conformation, N-methylation, tetra-substitution, -amino acids, and The attachment of a lipid moiety protected an unstable peptide side chain methylation(s). Unnatural amino acids are rarely the from enzymatic degradation [92]. The lipid was attached via either 4456 Current Medicinal Chemistry, 2012 Vol. 19, No. 26 Goodwin et al. substrates of proteolytic enzymes, therefore their inclusion into the (liposomes, micelles) carriers, a affect the release characteristics via sequence increased the stability of the modified peptide. For exam- diffusion through, and degradation of, the encapsulating ple, a D-peptide analogue of C-X-C chemokine receptor type 4 Fig. (7) [75, 124]. These particles effected bioadhesion (residence demonstrated dramatically improved stability. The natural L-peptide time and absorption), biodegradability (release kinetics), and bio- was degraded within 24 h, whereas the D-peptide showed no signs compatibility. Lanreotide, which has been used extensively in of degradation, even after 72 h [107]. The antimicrobial peptide Europe (approved by the FDA in 2007), was prepared by a micro- heptaibin primarily consisted of unnatural amino acids and was sphere encapsulation of biodegradable polymer to form a prolonged intact after 12 h treatment with proteolytic enzyme pronase E in release system [125]. Furthermore, nanoparticles were effective in Tris buffer [108]. Icatibant (Firazyr®, Shire), a bradykinin B2 delivering dalargin, an enkephalin analog, through the blood brain receptor antagonist decapeptide authorized by the European Com- barrier (BBB) [126]. Steric shielding proved successful for the pre- mission in 2008 and the FDA in 2011 for treatment of hereditary vention of peptide degradation on the surface of carrier molecules. angioedema, also contained unnatural amino acids [109]. D-amino A HIV cell-penetrating TAT peptide was shielded successfully acids have been used to make large oligomeric mimics of natural using PEG chains attached to the surface of micelles [127]. peptides. Using D-amino acids and synthesizing the peptide in re- Some peptides were used as vectors for enhanced transport. verse sequence order resulted in a similar side chain topology to the Delivery of the peptide dalargin to the brain was enhanced by the parent peptide. These “retro-inverso” (RI) peptides, exhibited simi- conjugation to the peptide vectors SynB1 and SynB3. These chi- lar binding activity with enhanced resistance to , there- meric peptides provided enhanced antinociception in mice and fore increased stability. A peptide with the potential for treatment of passed through the BBB by adsorptive-mediated endocytosis [128]. Alzheimer’s (OR2) was synthesized as an RI peptide. Taylor et al. demonstrated that the RI analogue remained intact after 24 h, by Conjugation to carrier molecules stabilized the structure of pep- which time the parent peptide has been completely degraded [110]. tides and/or acted as a vector to target specific cells/organs. For example, desmorphin coupled to the OX26 carrier or -endorphin The use of unnatural amino acids has enhanced interactions coupled to cationized albumin for delivery across the BBB [129, with peptide targets. D-Amino acid substitutions in -casomorphin 130]. Peptides have shown promise for use as a targeted anti-tumor were shown to enhance -opioid receptor binding and analgesia therapy. An anticancer drug, camptothecin, was conjugated to a [111]. A more complicated modification of a tumor-suppressing PEG carrier with an LHRH analogue (tumor targeting moiety). This p53-derived peptide, including the substitution of unnatural amino multifunctional polymer conferred enhanced antitumor activity over acids, led to a more stabilized helix, with an increase of affinity to the camptothecin-PEG conjugate alone [131]. Another peptide, IF7, the Hdm2 oncoprotein up to 63-fold [112]. targeted tumor vasculature within minutes, as demonstrated by conjugation to a florescent moiety and enhanced action after conju- Peptidomimetics gation to the anticancer drug, SN-38 [132]. In some cases, vectors In addition to the substitution of unnatural amino acids, modifi- have also been shown to lower the toxicity of cationic cytotoxic cations to peptide bonds improved peptide stability. Chemical peptides. An arginylglycylaspartic acid peptide recognizing v3 modifications to peptide bonds ranged from simple reductions, to integrin, which is overexpressed in tumour cells and vasculature, the replacement of the carbonyl or amide groups with esters, sul- coupled to a proapoptotic peptide killed tumor cells with lower fides and alkyls. This group of peptide analogues was classified as nonspecific cytoxicity than expected from cationic proapoptotic ‘peptidomimetics’ [113]. peptides [133]. Zuckermann and colleagues introduced a family of pepti- Cyclization domimetics called ‘’ [114]. The difference between pep- tides and peptoids was that the amino acid side chain shifted to the Cyclization of peptide sequences improved peptide stability by -amino group [115]. An antimicrobial ‘KLW’ peptide (Lys, Leu eliminating a degree of proteolysis. Since the 1940s, the interest in and Trp residues) was synthesized as a and was resistant to cyclic peptides has grown substantially [134]. One of the cyclic trypsin, without the loss of antimicrobial activity. The L-peptide, polypeptide drugs reported in 1945 was bacitracin, which was however, lost all activity after the enzymatic treatment [116]. Other recently approved by FDA for intramuscular treatment of infants important classes of oligomeric peptidomimetics include urea pep- with staphylococcal pneumonia. Depending on the functional tidomimetics, peptide-carbamates and peptido-sulfonamides Fig. groups, peptides were cyclized head-to-tail, head/tail-to-side-chain (6). Most peptidomimetics were synthesized using altered SPPS or side-chain-to-side-chain. Most commonly, cyclization was per- protocols [117-119]. Peptidomimetics and peptidomimetic-peptide formed by lactamization, lactonization and sulfide-based linkages. hybrids were used to alter peptide conformation by changing the Cyclization of herpes simplex virus by D, was shown bond rotations. Urea peptidomimetics and their hybrids mimicked to greatly increase enzymatic stability [135]. A thioether linkage the helical structure of host-defense peptides for use as antimicrobi- provided complete resistance to degradation in 50% human sera, als [120]. -Peptidomimetics also assumed defined helical struc- even after 96 h, while the linear peptide was completely degraded tures, with as few as six residues able to form a helix [121]. How- during this time. Cyclic variants including a disulfide bridge and ever, the properties of peptoids have conferred increased peptide bond were demonstrated to increase resistance to degrada- flexiblility-thus decreasing affinity-and sulfonamides have been tion with 29% and 73% of the peptide remaining, respectively shown to break -helical and -strand secondary structures [122]. [135]. Another example of natural bond modification was the substitu- Cyclization produced peptides with conformational restrictions. tion of disulfide bond(s) in cyclic peptides (e.g. -conotoxin). In Neurotoxin peptides (e.g. the conotoxins), somatostatin, oxytocin this case, selenocystein technology (based on the replacement of [123], LHRH [136] and insulin with sulfide-based bridges were all sulfur with selenium) was used to produce more stable and potent shown to be potent peptides. Ring closing metathesis (RCM) was nicotinic antagonists [123]. used in peptides, providing alkene staples as substitutes to sulfides and provided alternative constraints. Enkephalin [137], oxytocin Peptide Carrier/Delivery Systems [138], -conotoxin IMI [139] and lanreotide [140] were subjected to RCM modifications. Backbone cyclization of natural conotoxin In addition to increasing the stability of peptides, peptide carri- was shown to be effective for the development of an orally active ers or delivery systems such as microspheres, nanoparticles, PEG peptide for the treatment of neuropathic pain [141]. Stapled pep- and polyvinylpyrrolidone (PVP) polymers, along with lipid-based tides, such as inhibitors of induced myeloid leukemia cell different- Peptides As Therapeutics With Enhanced Bioactivity Current Medicinal Chemistry, 2012 Vol. 19, No. 26 4457

O R3 O R1 H H N N N N NATIVE PEPTIDE H H R4 O R2 O

O R3 O R1 O R3 O R1 H H H H N N D-AMINO ACID N N PARTIAL N N SUBSTITUTION N N RETRO-INVERSION H H H H R4 O R2 O R4 O R2 O

R3 R1 R1 O R3 O H H H H N N REDUCED PEPTIDE N N RETRO-INVERSO N N BOND N N H H H H PEPTIDE R4 R2 O R2 O R4

O R3 O R1 O R3 O R1 PEPTOID H H N N N N N AZAPEPTIDE N N N N H H R4 O R2 O R4 O R2 O R3 O R1 H H H H H UREA N N N N N -PEPTIDE N N N PEPTIDOMIMETIC H H H R3 O R2 O R1 O R2 O O R3 R5 O R1 O O R3 O O R2 O O R1 H PEPTIDO- N N N-MODIFIED PEPTIDE S S S SULFONAMIDE N N (e.g. methylation, N N H H H H hydroxylation) R4 O R2 O R5 R1 R4 O Me R3 Me R1 N O N OLIGO- N N METHYLATED & N O CARBAMATE N N REDUCED PEPTIDE BOND O R2 O R4 Me R2 Me R3 Fig. (6). Examples of peptidomimetics. Modifications range from simple reductions to the replacement of the carbonyl or amide groups with esters, sulfides and alkyls, as well as the use of D-confirmations and -amino acids.

Fig. (7). Conjugation and encapsulation of peptide therapeutics. A free peptide is degraded rapidly by proteolytic enzymes (A). A number of carrier-based transport systems block enzymes from binding to the peptide cargo and cleaving peptide bonds (B-D).

tiation protein, showed enhanced selectivity to the target [142]. pendent of a protein receptor and dependent on peptide oligomeri- Formed by simple “click chemistries”, 1,2,3-triazoles can mimic zation at the membrane surface [143]. trans- and cis-amide bonds, depending on substitutions. Cyclotides, Covalently “stapling” sections of the peptide chains enhanced cyclic plant peptides with three conserved disulfide-bonds, were stability in serum. For example, isomer A, the stapled -helix of found to be active against HIV. The cyclotide activity was inde- BH3 (previously shown to suppress leukemia cell growth), had 4458 Current Medicinal Chemistry, 2012 Vol. 19, No. 26 Goodwin et al. enhanced stability over the unmodified BID BH3 peptide in mouse tion techniques described herein will augment the potential of pep- serum [142]. The stapling of a tumor-suppressing, p53-derived tide therapeutics to come. peptide conferred a 4-fold increase over its wild-type [144]. Enfu- virtide was recently hydrocarbon stapled to remedy its instability. It CONFLICT OF INTEREST was suggested that these staples create a “proteolytic shield” by reinforcing an -helix and blocking proteolysis at the staple sites The author(s) confirm that this article content has no conflicts completely [145]. of interest.

Peptide Receptor and Transporter Targeting ACKNOWLEDGEMENTS Conjugation to molecules that are recognized by a transporter We thank the Australian Research Council for a Professorial or specific transport pathway facilitated transport across biological Research Fellowship to I.T. (DP110100212) and an Australian barriers. For example dermorphin (a natural opioid receptor ago- Postdoctoral Fellowship to P.S. (DP1092829). We also thank Ms nist), bound by a cleavable disulfide bridge to OX26 (a monoclonal Thalia Guerin for the critical review of the manuscript. antibody that binds the transferrin receptor), was shown to produce analgesia, confirming that it passed the BBB [129]. Further investi- REFERENCES gation identified the Na1-dependent glucose transporter was re- sponsible for the improved transportation of a glycosylated [1] Reichert, J.; Pechon, P.; Tartat, A.; Dunn, M.K. Development trends for tetrapeptide. The permeation of the -anomer was 5-times greater peptide therapeutics: A comprehensive quantitative analysis of peptide than that of the -conformation [77]. Glycosylation of Leu- therapeutics in clinical development.; Peptide Therapeutics Foundation: 2010. enkephalin at the N- or C-termini enhanced transport profiles in a [2] Bray, B.L. Large-scale manufacture of peptide therapeutics by chemical number of cases [102, 146, 147]. Addition of carbohydrates to the synthesis. Nat. Rev. Drug Discov., 2003, 2, 587-593. side chain of the serine on the N-terminus of the peptide was the [3] Thayer, A.M. Making peptides at large scale. Chem. Eng. News, 2011, 89, most successful modification for retaining its activity. Disaccharide 21-25. [4] Guzman, F.; Barberis, S.; Illanes, A. Peptide synthesis: Chemical or conjugates had considerable antinociceptive properties, with pro- enzymatic. Electron. J. Biotechnol., 2007, 10, 279-314. files similar to that of morphine [148]. [5] Du Vigneaud, V.; Ressler, C.; Swan, J.M.; Roberts, C.W.; Katsoyannis, P.G.; Gordon, S. The synthesis of an octapeptide amide with the hormonal activity Synthetic peptides were demonstrated by Stoermer et al. to bind of oxytocin. J. Am. Chem. Soc., 1953, 75, 4879-4880. to protease-activated G-protein coupled receptors. These short pep- [6] Merrifield, R.B. Solid phase peptide synthesis i. The synthesis of a tide sequences were of therapeutic interest because they activated tetrapeptide. J. Am. Chem. Soc., 1963, 85, 2149-2154. the receptor on immunoinflammatory and cancer cells, amongst [7] Schnolzer, M.; Alewood, P.; Jones, A.; Alewood, D.; Kent, S.B.H. In situ neutralization in boc-chemistry solid phase peptide synthesis - rapid, high others [149]. yield assembly of difficult sequences. Int. J. Pept. Res. Ther., 2007, 13, 31- Scanning, making libraries of peptides with substituted amino 44. [8] Chantell, C.A.; Onaiyekan, M.A.; Menakuru, M. Fast conventional Fmoc acids to determine the necessity and function of each bond and side solid-phase peptide synthesis: A comparative study of different activators. J. chain and potential cleavage sites, has led to the simplification and Pept. Sci., 2012, 18, 88-91. optimization of peptide chains. These methods generated analogues [9] Steinberg, M. Degarelix: A gonadotropin-releasing hormone antagonist for with improved physico-chemical characteristics. Scanning was the management of prostate cancer. Clin. Ther., 2009, 31, 2312-2331. D- [10] Verlander, M. Industrial applications of solid-phase peptide synthesis - a usually performed using (Ala-scan) [150] and amino status report. Int. J. Pept. Res. Ther., 2007, 13, 75-82. acids (D-scan) [151]. Ala-scans determined the residues important [11] Yu, H.M.; Chen, S.T.; Wang, K.T. Enhanced coupling efficiency in solid- for signaling and binding purposes and directed mutations were phase peptide-synthesis by microwave irradiation. J. Org. Chem., 1992, 57, performed to enhance affinity. An Ala-scan resolved the locations 4781-4784. for modifying the proinflammatory cytokine, IL-15. A simple [12] Rizzolo, F.; Sabatino, G.; Chelli, M.; Rovero, P.; Papini, A.M. A convenient microwave-enhanced solid-phase synthesis of difficult peptide sequences: amino acid substitution in the IL-1536-45 peptide led to a change in Case study of gramicidin a and CSF114(glc). Int. J. Pept. Res. Ther., 2007, the half maximal inhibitory concentration from 130M to 24M 13, 203-208. [152]. [13] Dawson, P.E.; Muir, T.W.; Clark-Lewis, I.; Kent, S.B. Synthesis of proteins by native chemical ligation. Science, 1994, 266, 776-779. [14] Offer, J.; Boddy, C.N.C.; Dawson, P.E. Extending synthetic access to CONCLUSIONS proteins with a removable acyl transfer auxiliary. J. Am. Chem. Soc., 2002, 124, 4642-4646. Along with biological aspects of developing peptides as thera- [15] Spasser, L.; Kumar, K.S.A.; Brik, A. Scope and limitation of side-chain peutics, manufacturing processes have been improved, making assisted ligation. J. Pept. Sci., 2011, 17, 252-255. [16] Hojo, K.; Maeda, M.; Kawasaki, K. Solid-phase peptide synthesis in water. peptides more attractive to the commercial market. A large number Part 3: A water-soluble N-protecting group, 2-[phenyl(methyl)sulfonio] of potential peptide therapeutics have not been FDA approved be- ethoxycarbonyl tetrafluoroborate, and its application to solid phase peptide cause of their poor physico-chemical characteristics, thus further synthesis in water. Tetrahedron, 2004, 60, 1875-1886. exploration of techniques aimed at improving the properties of po- [17] Hojo, K.; Ichikawa, H.; Onishi, M.; Fukumori, Y.; Kawasaki, K. Peptide synthesis 'in water' by a solution-phase method using water-dispersible tential peptide therapeutics is critical to the success of this field. nanoparticle Boc-amino acid. J. Pept. Sci., 2011, 17, 487-492. The addition of lipidic (e.g. lipoamino acids) and/or hydrophilic [18] Wohr, T.; Wahl, F.; Nefzi, A.; Rohwedder, B.; Sato, T.; Sun, X.C.; Mutter, units (e.g. carbohydrates, PEG) to potential peptide therapeutics M. Pseudo-prolines as a solubilizing, structure-disrupting protection represents an effective strategy for enhanced drug delivery in vivo, technique in peptide synthesis. J. Am. Chem. Soc., 1996, 118, 9218-9227. [19] Heinlein, C.; Silva, D.V.; Troster, A.; Schmidt, J.; Gross, A.; Unverzagt, C. especially for oral administration. The properties of lipids, carbo- Fragment condensation of C-terminal pseudoproline peptides without hydrates and polymers were exploited to render peptide sequences racemization on the solid phase. Angew. Chem. Int. Ed., 2011, 50, 6406- immunogenic, resulting in novel peptide-based vaccine systems. 6410. Polymers were also extensively researched for the delivery of pep- [20] Frutos, S.; Tulla-Puche, J.; Albericio, F.; Giralt, E. Chemical synthesis of F- 19-labeled HIV-1 protease using Fmoc-chemistry and ChemMatrix resin. Int. tides, either by their attachment or encapsulation. The use of pepti- J. Pept. Res. Ther., 2007, 13, 221-227. domimetics, unnatural amino acids and cyclization technologies [21] Garcia-Martin, F.; Quintanar-Audelo, M.; Garcia-Ramos, Y.; Cruz, L.J.; provided peptides with enhanced characteristics. With the promis- Gravel, C.; Furic, R.; Cote, S.; Tulla-Puche, J.; Albericio, F. ChemMatrix, a ing in vitro and in vivo results that all of these delivery systems poly(ethylene glycol)-based support for the solid-phase synthesis of complex peptides. J. Comb. Chem., 2006, 8, 213-220. displayed, many of these analogues and conjugates will progress [22] Garcia-Martin, F.; White, P.; Steinauer, R.; Cote, S.; Tulla-Puche, J.; into clinical trials. It is foreseeable that the synthesis and modifica- Albericio, F. The synergy of ChemMatrix resin (R) and pseudoproline Peptides As Therapeutics With Enhanced Bioactivity Current Medicinal Chemistry, 2012 Vol. 19, No. 26 4459

building blocks renders RANTES, a complex aggregated chemokine. Wiesmuller, K.H.; Jung, G. Bacterial lipopeptides constitute efficient novel Biopolymers, 2006, 84, 566-575. immunogens and adjuvants in parenteral and oral immunization. Behring [23] Kassem, T.; Sabatino, D.; Jia, X.; Zhu, X.X.; Lubell, W.D. To rink or not to Inst. Mitt., 1997, 390-399. rink amide link, that is the question to address for more economical and [52] Andrieu, M.; Loing, E.; Desoutter, J.F.; Connan, F.; Choppin, J.; Gras- environmentally sound solid-phase peptide synthesis. Int. J. Pept. Res. Ther., Masse, H.; Hanau, D.; Dautry-Varsat, A.; Guillet, J.G.; Hosmalin, A. 2009, 15, 211-218. Endocytosis of an HIV-derived lipopeptide into human dendritic cells [24] Agyei, D.; Danquah, M.K. Industrial-scale manufacturing of pharmaceutical- followed by class I-restricted CD8(+) T lymphocyte activation. Eur. J. grade bioactive peptides. Biotechnol. Adv., 2011, 29, 272-277. Immunol., 2000, 30, 3256-3265. [25] Agathos, S.N.; Madhosingh, C.; Marshall, J.W.; Lee, J. The fungal [53] Phillipps, K.S.M.; Wykes, M.N.; Liu, X.Q.; Brown, M.; Blanchfield, J.; production of cyclosporine. Ann. N. Y. Acad. Sci., 1987, 506, 657-662. Toth, I. A novel synthetic adjuvant enhances dendritic cell function. [26] Oldenburg, K.R.; D'Orfani, A.L.; Selick, H.E. A method for the high-level Immunology, 2009, 128, e582-e588. expression of a analog in Escherichia coli. Protein [54] Olive, C.; Batzloff, M.R.; Toth, I. Lipid core peptide technology and group A Expression Purif., 1994, 5, 278-284. streptococcal vaccine delivery. Expert Rev. Vaccines, 2004, 3, 43-58. [27] Roy, S.K. Clinical Pharmacology and Biopharmaceutics Review - Desirudin; [55] Olive, C.; Ho, M.F.; Dyer, J.; Lincoln, D.; Barozzi, N.; Toth, I.; Good, M.F. U.S. Food and Drug Administration, 2000. Immunization with a tetraepitopic lipid core peptide vaccine construct [28] Forteo(r) teriparatide (rdna origin) injection; Eli Lilly and Company: induces broadly protective immune responses against group A streptococcus. Indianapolis, IN 46285, USA, 2002, 2007; pp 1-18. J. Infect. Dis., 2006, 193, 1666-1676. [29] Lehmann, A. Ecallantide (dx-88), a plasma kallikrein inhibitor for the [56] Zhong, G.M.; Toth, I.; Reid, R.; Brunham, R.C. Immunogenicity evaluation treatment of hereditary angioedema and the prevention of blood loss in on- of a lipidic amino acid-based synthetic peptide vaccine for chlamydia- pump cardiothoracic surgery. Expert Opin. Biol. Ther., 2008, 8, 1187-1199. trachomatis. J. Immunol., 1993, 151, 3728-3736. [30] Won, J.I.; Barron, A.E. A new cloning method for the preparation of long [57] Moyle, P.M.; Olive, C.; Ho, M.F.; Pandey, M.; Dyer, J.; Suhrbier, A.; Fujita, repetitive polypeptides without a sequence requirement. Macromolecules, Y.; Toth, I. Toward the development of prophylactic and therapeutic human 2002, 35, 8281-8287. papillomavirus type-16 lipopeptide vaccines. J. Med. Chem., 2007, 50, 4721- [31] Martemyanov, K.A.; Shirokov, V.A.; Kurnasov, O.V.; Gudkov, A.T.; Spirin, 4727. A.S. Cell-free production of biologically active polypeptides: Application to [58] Olive, C.; Schulze, K.; Sun, H.K.; Ebensen, T.; Horvath, A.; Toth, I.; the synthesis of antibacterial peptide cecropin. Protein Expression Purif., Guzman, C.A. Enhanced protection against Streptococcus pyogenes 2001, 21, 456-461. by intranasal vaccination with a dual antigen component M protein/SfbI lipid [32] Breitling, R.; Klingner, S.; Callewaert, N.; Pietrucha, R.; Geyer, A.; Ehrlich, core peptide vaccine formulation. Vaccine, 2007, 25, 1789-1797. G.; Hartung, R.; Muller, A.; Contreras, R.; Beverley, S.M.; Alexandrov, K. [59] Simerska, P.; Abdel-Aal, A.B.; Fujita, Y.; Batzloff, M.R.; Good, M.F.; Toth, Non-pathogenic trypanosomatid protozoa as a platform for protein research I. Synthesis and in vivo studies of carbohydrate-based vaccines against group and production. Protein Expression Purif., 2002, 25, 209-218. A streptococcus. Biopolymers, 2008, 90, 611-616. [33] Mureev, S.; Kovtun, O.; Nguyen, U.T.T.; Alexandrov, K. Species- [60] Moyle, P.M.; Olive, C.; Ho, M.F.; Good, M.F.; Toth, I. Synthesis of a highly independent translational leaders facilitate cell-free expression. Nat. pure lipid core peptide based self-adjuvanting triepitopic group A Biotechnol., 2009, 27, 747-798. streptococcal vaccine, and subsequent immunological evaluation. J. Med. [34] Wang, K.; Schmied, W.H.; Chin, J.W. Reprogramming the : Chem., 2006, 49, 6364-6370. From triplet to quadruplet codes. Angew. Chem. Int. Ed., 2012, 51, 2288- [61] Simerska, P.; Abdel-Aal, A.B.; Fujita, Y.; Moyle, P.M.; McGeary, R.P.; 2297. Batzloff, M.R.; Olive, C.; Good, M.F.; Toth, I. Development of a [35] Neumann, H.; Wang, K.H.; Davis, L.; Garcia-Alai, M.; Chin, J.W. Encoding liposaccharide-based delivery system and its application to the design of multiple unnatural amino acids via evolution of a quadruplet-decoding group A streptococcal vaccines. J. Med. Chem., 2008, 51, 1447-1452. . Nature, 2010, 464, 441-444. [62] Abdel-Aal, A.B.M.; Zaman, M.; Fujita, Y.; Batzloff, M.R.; Good, M.F.; [36] Murray, B.A.; FitzGerald, R.J. Angiotensin converting enzyme inhibitory Toth, I. Design of three-component vaccines against group A streptococcal peptides derived from food proteins: Biochemistry, bioactivity and : Importance of spatial arrangement of vaccine components. J. production. Curr. Pharm. Des., 2007, 13, 773-791. Med. Chem., 2010, 53, 8041-8046. [37] Gill, H.S.; Doull, F.; Rutherfurd, K.J.; Cross, M.L. Immunoregulatory [63] Olive, C.; Moyle, P.M.; Toth, I. Towards the development of a broadly peptides in bovine milk. Br. J. Nutr., 2000, 84 Suppl 1, S111-117. protective group A streptococcal vaccine based on the lipid-core peptide [38] Hartmann, R.; Meisel, H. Food-derived peptides with biological activity: system. Curr. Med. Chem., 2007, 14, 2976-2988. From research to food applications. Curr. Opin. Biotechnol., 2007, 18, 163- [64] Adams, E.W.; Ratner, D.M.; Seeberger, P.H.; Hacohen, N. Carbohydrate- 169. mediated targeting of antigen to dendritic cells leads to enhanced [39] Bourgeaux, V.; Cadene, M.; Piller, F.; Piller, V. Efficient enzymatic presentation of antigen to T cells. ChemBioChem, 2008, 9, 294-303. glycosylation of peptides and oligosaccharides from GalNAc and UTP. [65] Cui, Z.G.; Han, S.J.; Huang, L. Coating of mannan on LPD particles ChemBioChem, 2007, 8, 37-40. containing HPV E7 peptide significantly enhances immunity against HPV- [40] Merkler, D.J. C-terminal amidated peptides - production by the in-vitro positive tumor. Pharm. Res., 2004, 21, 1018-1025. enzymatic amidation of -extended peptides and the importance of the [66] Skwarczynski, M.; Toth, I. Peptide-based subunit nanovaccines. Curr. Drug amide to bioactivity. Enzyme Microb. Technol., 1994, 16, 450-456. Del., 2011, 8, 282-289. [41] Knudsen, L.B.; Nielsen, P.F.; Huusfeldt, P.O.; Johansen, N.L.; Madsen, K.; [67] Zaman, M.; Skwarczynski, M.; Malcolm, J.M.; Urbani, C.N.; Jia, Z.F.; Pedersen, F.Z.; Thogersen, H.; Wilken, M.; Agerso, H. Potent derivatives of Batzloff, M.R.; Good, M.F.; Monteiro, M.J.; Toth, I. Self-adjuvanting glucagon-like peptide-1 with pharmacokinetic properties suitable for once polyacrylic nanoparticulate delivery system for group A streptococcus (gas) daily administration. J. Med. Chem., 2000, 43, 1664-1669. vaccine. Nanomed. Nanotechnol. Biol. Med., 2011, 7, 168-173. [42] Drucker, D.J.; Drutselis, A.; Kirkpatrick, P. Fresh from the pipeline. [68] Skwarczynski, M.; Zaman, M.; Urbani, C.N.; Lin, I.C.; Jia, Z.F.; Batzloff, Liraglutide. Nat. Rev. Drug Discov., 2010, 9, 267-268. M.R.; Good, M.F.; Monteiro, M.F.; Toth, I. Polyacrylate dendrimer [43] Leadbetter, M.R.; Adams, S.M.; Bazzini, B.; Fatheree, P.R.; Karr, D.E.; nanoparticles: A self-adjuvanting vaccine delivery system. Angew. Chem. Krause, K.M.; Lam, B.M.; Linsell, M.S.; Nodwell, M.B.; Pace, J.L.; Quast, Int. Ed., 2010, 49, 5742-5745. K.; Shaw, J.P.; Soriano, E.; Trapp, S.G.; Villena, J.D.; Wu, T.X.; [69] McNeela, E.A.; Lavelle, E.C. Recent advances in microparticle and Christensen, B.G.; Judice, J.K. Hydrophobic vancomycin derivatives with nanoparticle delivery vehicles for mucosal vaccination. Curr. Top. improved ADME properties: Discovery of telavancin (TD-6424). The Microbiol. Immunol., 2012, 354, 75-99. Journal of Antibiotics, 2004, 57, 326-336. [70] Woodley, J.F. Enzymatic barriers for GI peptide and protein delivery. Crit. [44] Babiuk, L.A. Broadening the approaches to developing more effective Rev. Ther. Drug Carrier Syst., 1994, 11, 61-95. vaccines. Vaccine, 1999, 17, 1587-1595. [71] Kawakami, T.; Kamo, M.; Takamoto, K.; Miyazaki, K.; Chow, L.P.; Ueno, [45] Moyle, P.M.; Toth, I. Self-adjuvanting lipopeptide vaccines. Curr. Med. Y.; Tsugita, A. Bond-specific chemical cleavages of peptides and proteins Chem., 2008, 15, 506-516. with perfluoric acid vapors: Novel peptide bond cleavages of glycyl- [46] Moyle, P.M.; McGeary, R.P.; Blanchfield, J.T.; Toth, I. Mucosal , the amino side of serine residues and the carboxyl side of aspartic immunisation: Adjuvants and delivery systems. Curr. Drug Del., 2004, 1, acid residues. J. Biochem., 1997, 121, 68-76. 385-396. [72] Vlieghe, P.; Lisowski, V.; Martinez, J.; Khrestchatisky, M. Synthetic [47] Tam, J.P. Synthetic peptide vaccine design: Synthesis and properties of a therapeutic peptides: Science and market. Drug Discov. Today, 2010, 15, 40- high-density multiple antigenic peptide system. Proc. Natl. Acad. Sci. U. S. 56. A., 1988, 85, 5409-5413. [73] Barberi-Heyob, M.; Pernot, M.; Vanderesse, R.; Frochot, C.; Guillemin, F. [48] Purcell, A.W.; Croft, N.P. Peptidomimetics: Modifying peptides in the Stability of peptides and therapeutic success in cancer. Expert Opin. Drug pursuit of better vaccines. Expert Rev. Vaccines, 2011, 10, 211-226. Metab. Toxicol., 2011, 7, 793-802. [49] Wiesmuller, K.H.; Jung, G.; Hess, G. Novel low-molecular-weight synthetic [74] Nguyen, L.T.; Chau, J.K.; Perry, N.A.; de Boer, L.; Zaat, S.A.J.; Vogel, H.J. vaccine against foot-and-mouth disease containing a potent B-cell and Serum stabilities of short -and -rich antimicrobial peptide macrophage activator. Vaccine, 1989, 7, 29-33. analogs. PLoS ONE, 2010, 5, e12684. doi:10.1371/journal.pone.0012684. [50] Zeng, W.G.; Ghosh, S.; Lau, Y.F.; Brown, L.E.; Jackson, D.C. Highly [75] Zhang, G.H.; Han, B.Z.; Lin, X.Y.; Wu, X.; Yan, H.S. Modification of immunogenic and totally synthetic lipopeptides as self-adjuvanting antimicrobial peptide with low molar mass poly(ethylene glycol). J. immunocontraceptive vaccines. J. Immunol., 2002, 169, 4905-4912. Biochem., 2008, 144, 781-788. [51] Bessler, W.G.; Baier, W.; vd Esche, U.; Hoffmann, P.; Heinevetter, L.; [76] Bergeon, J.A.; Chan, Y.N.; Charles, B.G.; Toth, I. Oral absorption 4460 Current Medicinal Chemistry, 2012 Vol. 19, No. 26 Goodwin et al.

enhancement of dipeptide l-Glu-l-Trp-OH by lipid and glycosyl conjugation. potential antinociceptive agents. Int. J. Pharm., 1998, 161, 55-64. Biopolymers, 2008, 90, 633-643. [103] Drouillat, B.; Hillery, A.M.; Dekany, G.; Falconer, R.; Wright, K.; Toth, I. [77] Nomoto, M.; Yamada, K.; Haga, M.; Hayashi, M. Improvement of intestinal Novel liposaccharide conjugates for drug and peptide delivery. J. Pharm. absorption of peptide drugs by glycosylation: Transport of tetrapeptide by the Sci., 1998, 87, 25-30. sodium ion-dependent D-glucose transporter. J. Pharm. Sci., 1998, 87, 326- [104] Wessling, S.T.; Ross, B.P.; Koda, Y.; Blanchfield, J.T.; Toth, I. CACO-2 cell 332. permeability and stability of two D-glucopyranuronamide conjugates of [78] Egleton, R.D.; Mitchell, S.A.; Huber, J.D.; Palian, M.M.; Polt, R.; Davis, thyrotropin-releasing hormone. Biorg. Med. Chem., 2007, 15, 4946-4950. T.P. Improved blood-brain barrier penetration and enhanced analgesia of an [105] Toth, I.; Malkinson, J.P.; Flinn, N.S.; Drouillat, B.; Horvath, A.; Erchegyi, J.; opioid peptide by glycosylation. J. Pharmacol. Exp. Ther., 2001, 299, 967- Idei, M.; Venetianer, A.; Artursson, P.; Lazorova, L.; Szende, B.; Keri, G. 972. Novel lipoamino acid- and liposaccharide-based system for peptide delivery: [79] Foldvari, M.; Baca-Estrada, M.E.; He, Z.H.; Hu, J.P.; Attah-Poku, S.; King, Application for oral administration of tumor-selective somatostatin M. Dermal and transdermal delivery of protein pharmaceuticals: Lipid-based analogues. J. Med. Chem., 1999, 42, 4010-4013. delivery systems for interferon alpha. Biotechnol. Appl. Biochem., 1999, 30, [106] Suzuki, K.; Susaki, H.; Okuno, S.; Yamada, H.; Watanabe, H.K.; Sugiyama, 129-137. Y. Specific renal delivery of sugar-modified low-molecular-weight peptides. [80] Wollmer, P.; Pieber, T.R.; Gall, M.A.; Brunton, S. Delivering needle-free J. Pharmacol. Exp. Ther., 1999, 288, 888-897. insulin using AERx iDMS (insulin diabetes management system) [107] Zhou, N.M.; Luo, Z.W.; Luo, J.S.; Fan, X.J.; Cayabyab, M.; Hiraoka, M.; technology. Diabetes Technol. Ther., 2007, 9 Suppl 1, S57-64. Liu, D.X.; Han, X.B.; Pesavento, J.; Dong, C.Z.; Wang, Y.L.; An, J.; Kaji, [81] Hamman, J.H.; Enslin, G.M.; Kotze, A.F. Oral delivery of peptide drugs - H.; Sodroski, J.G.; Huang, Z.W. Exploring the stereochemistry of CXCR4- barriers and developments. BioDrugs, 2005, 19, 165-177. peptide recognition and inhibiting HIV-1 entry with D-peptides derived from [82] Huzil, J.T.; Sivaloganathan, S.; Kohandel, M.; Foldvari, M. Drug delivery chemokines. J. Biol. Chem., 2002, 277, 17476-17485. through the skin: Molecular simulations of barrier lipids to design more [108] De Zotti, M.; Biondi, B.; Peggion, C.; Park, Y.; Hahm, K.S.; Formaggio, F.; effective noninvasive dermal and transdermal delivery systems for small Toniolo, C. Synthesis, preferred conformation, protease stability, and molecules, biologics, and cosmetics. Wiley Interdiscip. Rev. Nanomed. membrane activity of heptaibin, a medium-length peptaibiotic. J. Pept. Sci., Nanobiotechnol., 2011, 3, 449-462. 2011, 17, 585-594. [83] Chen, X.; Fernando, G.J.P.; Crichton, M.L.; Flaim, C.; Yukiko, S.R.; [109] Bork, K.; Yasothan, U.; Kirkpatrick, P. Fresh from the pipeline. Icatibant. Fairmaid, E.J.; Corbett, H.J.; Primiero, C.A.; Ansaldo, A.B.; Frazer, I.H.; Nat. Rev. Drug Discov., 2008, 7, 801-802. Brown, L.E.; Kendall, M.A.F. Improving the reach of vaccines to low- [110] Taylor, M.; Moore, S.; Mayes, J.; Parkin, E.; Beeg, M.; Canovi, M.; Gobbi, resource regions, with a needle-free vaccine delivery device and long-term M.; Mann, D.M.A.; Allsop, D. Development of a proteolytically stable retro- thermostabilization. J. Controlled Release, 2011, 152, 349-355. inverso peptide inhibitor of beta-amyloid oligomerization as a potential novel [84] Scheuch, G.; Siekmeier, R. Novel approaches to enhance pulmonary delivery treatment for alzheimer's disease. Biochemistry (Mosc). 2010, 49, 3261-3272. of proteins and peptides. J. Physiol. Pharmacol., 2007, 58, 615-625. [111] Liebmann, C.; Szucs, M.; Neubert, K.; Hartrodt, B.; Arold, H.; Barth, A. [85] Bailey, C.J.; Barnett, A.H. Why is Exubera being withdrawn? Br. Med. J., Opiate receptor-binding affinities of some D-amino-acid substituted beta- 2007, 335, 1156-1156. casomorphin analogs. Peptides, 1986, 7, 195-199. [86] Chung, S.W.; Hil-lal, T.A.; Byun, Y. Strategies for non-invasive delivery of [112] Garcia-Echeverria, C.; Chene, P.; Blommers, M.J.J.; Furet, P. Discovery of biologics. J. Drug Targeting, 2012, 20, 481-501. potent antagonists of the interaction between human double minute 2 and [87] du Plessis, L.H.; Kotze, A.F.; Junginger, H.E. Nasal and rectal delivery of tumor suppressor p53. J. Med. Chem., 2000, 43, 3205-3208. insulin with chitosan and N-trimethyl chitosan chloride. Drug Deliv., 2010, [113] Liskamp, R.M.J.; Rijkers, D.T.S.; Kruijtzer, J.A.W.; Kemmink, J. Peptides 17, 399-407. and proteins as a continuing exciting source of inspiration for [88] Zhang, X.G.; Zhang, H.J.; Wu, Z.M.; Wang, Z.; Niu, H.M.; Li, C.X. Nasal peptidomimetics. ChemBioChem, 2011, 12, 1626-1653. absorption enhancement of insulin using PEG-grafted chitosan nanoparticles. [114] Zuckermann, R.N.; Kerr, J.M.; Kent, S.B.H.; Moos, W.H. Efficient method Eur. J. Pharm. Biopharm., 2008, 68, 526-534. for the preparation of peptoids [oligo(N-substituted )] by [89] Dufes, C.; Olivier, J.C.; Gaillard, F.; Gaillard, A.; Couet, W.; Muller, J.M. submonomer solid-phase synthesis. J. Am. Chem. Soc., 1992, 114, 10646- Brain delivery of vasoactive intestinal peptide (VIP) following nasal 10647. administration to rats. Int. J. Pharm., 2003, 255, 87-97. [115] Miller, S.M.; Simon, R.J.; Ng, S.; Zuckermann, R.N.; Kerr, J.M.; Moos, [90] Antosova, Z.; Mackova, M.; Kral, V.; Macek, T. Therapeutic application of W.H. Comparison of the proteolytic susceptibilities of homologous L-amino- peptides and proteins: Parenteral forever? Trends Biotechnol., 2009, 27, 628- acid, D-amino-acid, and N-substituted glycine peptide and peptoid 635. oligomers. Drug Dev. Res., 1995, 35, 20-32. [91] Rose, E.H.; Aledort, L.M. Nasal spray desmopressin (DDAVP) for mild [116] Bang, J.K.; Nan, Y.H.; Lee, E.K.; Shin, S.Y. A novel Trp-rich model hemophilia A and von Willebrand disease. Ann. Intern. Med., 1991, 114, antimicrobial peptoid with increased protease stability. Bull. Korean Chem. 563-568. Soc., 2010, 31, 2509-2513. [92] Toth, I.; Flinn, N.; Hillery, A.; Gibbons, W.A.; Artursson, P. Lipidic [117] Kim, J.M.; Bi, Y.Z.; Paikoff, S.J.; Schultz, P.G. The solid phase synthesis of conjugates of luteinizing-hormone-releasing hormone (LHRH)+ and oligoureas. Tetrahedron Lett., 1996, 37, 5305-5308. thyrotropin-releasing-hormone (TRH)+ that release and protect the native [118] Cho, C.Y.; Liu, C.W.; Wemmer, D.E.; Schultz, P.G. Cyclic and linear in homogenates of human intestinal epithelial (CACO-2) cells. Int. oligocarbamate ligands for human thrombin. Biorg. Med. Chem., 1999, 7, J. Pharm., 1994, 105, 241-247. 1171-1179. [93] Bundgaard, H.; Hansen, A.B. Pro-drugs as drug delivery systems. Pharm. [119] deBont, D.B.A.; Moree, W.J.; Liskamp, R.M.J. Molecular diversity of Int., 1981, 2, 136-140. peptidomimetics: Approaches to the solid-phase synthesis of [94] Testa, B. Prodrugs: Bridging pharmacodynamic/pharmacokinetic gaps. Curr. peptidosulfonamides. Biorg. Med. Chem., 1996, 4, 667-672. Opin. Chem. Biol., 2009, 13, 338-344. [120] Claudon, P.; Violette, A.; Lamour, K.; Decossas, M.; Fournel, S.; Heurtault, [95] Hsieh, P.W.; Hung, C.F.; Fang, J.Y. Current prodrug design for drug B.; Godet, J.; Mely, Y.; Jamart-Gregoire, B.; Averlant-Petit, M.C.; Briand, discovery. Curr. Pharm. Des., 2009, 15, 2236-2250. J.P.; Duportail, G.; Monteil, H.; Guichard, G. Consequences of isostructural [96] Blanchfield, J.T.; Lew, R.A.; Smith, A.I.; Toth, I. The stability of lipidic main-chain modifications for the design of antimicrobial foldamers: Helical analogues of GnRH in plasma and kidney preparations: The stereoselective mimics of host-defense peptides based on a heterogeneous amide/urea release of the parent peptide. Bioorg. Med. Chem. Lett., 2005, 15, 1609-1612. backbone. Angew. Chem. Int. Ed., 2010, 49, 333-336. [97] Ferdinandi, E.S.; Brazeau, P.; High, K.; Procter, B.; Fennell, S.; Dubreuil, P. [121] Seebach, D.; Overhand, M.; Kuhnle, F.N.M.; Martinoni, B.; Oberer, L.; Non-clinical pharmacology and safety evaluation of TH9507, a human Hommel, U.; Widmer, H. Beta-peptides: Synthesis by Arndt-Eistert -releasing factor analogue. Basic Clin. Pharmacol. Toxicol., homologation with concomitant peptide coupling. Structure determination by 2007, 100, 49-58. NMR and CD spectroscopy and by X-ray crystallography. Helical secondary [98] Koda, Y.; Liang, M.T.; Blanchfield, J.T.; Toth, I. In vitro stability and structure of a beta-hexapeptide in solution and its stability towards pepsin. permeability studies of liposomal delivery systems for a novel lipophilic Helv. Chim. Acta, 1996, 79, 913-941. endomorphin 1 analogue. Int. J. Pharm., 2008, 356, 37-43. [122] Elgersma, R.C.; Meijneke, T.; de Jong, R.; Brouwer, A.J.; Posthuma, G.; [99] Toth, I.; Hillery, A.M.; Wood, I.P.; Magnusson, C.; Artursson, P. Oral Rijkers, D.T.S.; Liskamp, R.M.J. Synthesis and structural investigations of absorption of lipidic amino-acid conjugates. Int. J. Pharm., 1994, 102, 223- N-alkylated beta-peptidosulfonamide-peptide hybrids of the amyloidogenic 230. amylin(20-29) sequence: Implications of supramolecular folding for the [100] Uchiyama, T.; Kotani, A.; Tatsumi, H.; Kishida, T.; Okamoto, A.; Okada, design of peptide-based bionanomaterials. Org. Biomol. Chem., 2006, 4, N.; Murakami, M.; Fujita, T.; Fujiwara, Y.; Kiso, Y.; Muranishi, S.; 3587-3597. Yamamoto, A. Development of novel lipophilic derivatives of DADLE [123] Muttenthaler, M.; Andersson, A.; de Araujo, A.D.; Dekan, Z.; Lewis, R.J.; ( enkephalin analogue): Intestinal permeability characteristics of Alewood, P.F. Modulating oxytocin activity and plasma stability by disulfide DADLE derivatives in rats. Pharm. Res., 2000, 17, 1461-1467. bond engineering. J. Med. Chem., 2010, 53, 8585-8596. [101] Fredholt, K.; Adrian, C.; Just, L.; Larsen, D.H.; Weng, S.S.; Moss, B.; Friis, [124] Malik, D.K.; Baboota, S.; Ahuja, A.; Hasan, S.; Ali, J. Recent advances in G.J. Chemical and enzymatic stability as well as transport properties of a leu- protein and peptide drug delivery systems. Curr. Drug Del., 2007, 4, 141- enkephalin analogue and ester prodrugs thereof. J. Controlled Release, 2000, 151. 63, 261-273. [125] Toledano, Y.; Rot, L.; Greenman, Y.; Orlovsky, S.; Pauker, Y.; Olchovsky, [102] Kellam, B.; Drouillat, B.; Dekany, G.; Starr, M.S.; Toth, I. Synthesis and in D.; Eliash, A.; Bardicef, O.; Makhoul, O.; Tsvetov, G.; Gershinsky, M.; vitro evaluation of lipoamino acid and carbohydrate-modified enkephalins as Cohen-Ouaqnine, O.; Ness-Abramof, R.; Adnan, Z.; Ilany, J.; Guttmann, H.; Peptides As Therapeutics With Enhanced Bioactivity Current Medicinal Chemistry, 2012 Vol. 19, No. 26 4461

Sapir, M.; Benbassat, C.; Shimon, I. Efficacy of long-term lanreotide Sci., 2007, 13, 280-285. treatment in patients with . Pituitary, 2009, 12, 285-293. [140] Whelan, A.N.; Elaridi, J.; Mulder, R.J.; Robinson, A.J.; Jackson, W.R. [126] Alyautdin, R.; Gothier, D.; Petrov, V.; Kharkevich, D.; Kreuter, J. Analgesic Metal-catalysed tandem metathesis-hydrogenation reactions for the synthesis activity of the hexapeptide dalargin adsorbed on the surface of polysorbate of carba analogues of cyclic peptides. Can. J. Chem., 2005, 83, 875-881. 80-coated poly(butyl cyanoacrylate) nanoparticles. Eur. J. Pharm. [141] Clark, R.J.; Akcan, M.; Kaas, Q.; Daly, N.L.; Craik, D.J. Cyclization of Biopharm., 1995, 41, 44-48. conotoxins to improve their biopharmaceutical properties. Toxicon, 2012, 59, [127] Koren, E.; Apte, A.; Sawant, R.R.; Grunwald, J.; Torchilin, V.P. Cell- 446-455. penetrating TAT peptide in drug delivery systems: Proteolytic stability [142] Walensky, L.D.; Kung, A.L.; Escher, I.; Malia, T.J.; Barbuto, S.; Wright, requirements. Drug Deliv., 2011, 18, 377-384. R.D.; Wagner, G.; Verdine, G.L.; Korsmeyer, S.J. Activation of apoptosis in [128] Rousselle, C.; Clair, P.; Smirnova, M.; Kolesnikov, Y.; Pasternak, G.W.; vivo by a hydrocarbon-stapled BH3 helix. Science, 2004, 305, 1466-1470. Gac-Breton, S.; Rees, A.R.; Scherrmann, J.M.; Temsamani, J. Improved [143] Sando, L.; Henriques, S.T.; Foley, F.; Simonsen, S.M.; Daly, N.L.; Hall, brain uptake and pharmacological activity of dalargin using a peptide-vector- K.N.; Gustafson, K.R.; Aguilar, M.I.; Craik, D.J. A synthetic mirror image of mediated strategy. J. Pharmacol. Exp. Ther., 2003, 306, 371-376. kalata b1 reveals that cyclotide activity is independent of a protein receptor. [129] Bickel, U.; Kang, Y.S.; Pardridge, W.M. In vivo cleavability of a disulfide- ChemBioChem, 2011, 12, 2456-2462. based chimeric opioid peptide in rat brain. Bioconj. Chem., 1995, 6, 211-218. [144] Bernal, F.; Tyler, A.F.; Korsmeyer, S.J.; Walensky, L.D.; Verdine, G.L. [130] Kumagai, A.K.; Eisenberg, J.B.; Pardridge, W.M. Absorptive-mediated Reactivation of the p53 tumor suppressor pathway by a stapled p53 peptide. endocytosis of cationized albumin and a beta-endorphin-cationized albumin J. Am. Chem. Soc., 2007, 129, 5298-5298. chimeric peptide by isolated brain capillaries - model system of blood-brain- [145] Bird, G.H.; Madani, N.; Perry, A.F.; Princiotto, A.M.; Supko, J.G.; He, X.Y.; barrier transport. J. Biol. Chem., 1987, 262, 15214-15219. Gavathiotis, E.; Sodroski, J.G.; Walensky, L.D. Hydrocarbon double-stapling [131] Chandna, P.; Khandare, J.J.; Ber, E.; Rodriguez-Rodriguez, L.; Minko, T. remedies the proteolytic instability of a lengthy peptide therapeutic. Proc. Multifunctional tumor-targeted polymer-peptide-drug delivery system for Natl. Acad. Sci. U. S. A., 2010, 107, 14093-14098. treatment of primary and metastatic cancers. Pharm. Res., 2010, 27, 2296- [146] Wong, A.K.; Ross, B.P.; Chan, Y.N.; Artursson, P.; Lazorova, L.; Jones, A.; 2306. Toth, I. Determination of transport in the CACO-2 cell assay of compounds [132] Hatakeyama, S.; Sugihara, K.; Shibata, T.K.; Nakayama, J.; Akama, T.O.; varying in lipophilicity using LC-MS: Enhanced transport of leu-enkephalin Tamura, N.; Wong, S.M.; Bobkov, A.A.; Takano, Y.; Ohyama, C.; Fukuda, analogues. Eur. J. Pharm. Sci., 2002, 16, 113-118. M.; Fukuda, M.N. Targeted drug delivery to tumor vasculature by a [147] Wu, S.; Campbell, C.; Koda, Y.; Blanchfield, J.T.; Toth, I. Investigation of carbohydrate mimetic peptide. Proc. Natl. Acad. Sci. U. S. A., 2011, 108, the route of absorption of lipid and sugar modified leu-enkephalin analogues 19587-19592. and their enzymatic stability using the CACO-2 cell monolayer system. Med. [133] Dufort, S.; Sancey, L.; Hurbin, A.; Foillard, S.; Boturyn, D.; Dumy, P.; Coll, Chem., 2006, 2, 203-211. J.L. Targeted delivery of a proapoptotic peptide to tumors in vivo. J. Drug [148] Elmagbari, N.O.; Egleton, R.D.; Palian, M.M.; Lowery, J.J.; Schmid, W.R.; Targeting, 2011, 19, 582-588. Davis, P.; Navratilova, E.; Dhanasekaran, M.; Keyari, C.M.; Yamamura, [134] White, C.J.; Yudin, A.K. Contemporary strategies for peptide H.I.; Porreca, F.; Hruby, V.J.; Polt, R.; Bilsky, E.J. Antinociceptive structure- macrocyclization. Nat. Chem., 2011, 3, 509-524. activity studies with enkephalin-based opioid . J. Pharmacol. [135] Tugyi, R.; Mezo, G.; Fellinger, E.; Andreu, D.; Hudecz, F. The effect of Exp. Ther., 2004, 311, 290-297. cyclization on the enzymatic degradation of herpes simplex virus [149] Stoermer, M.J.; Flanagan, B.; Beyer, R.L.; Madala, P.K.; Fairlie, D.P. glycoprotein d derived epitope peptide. J. Pept. Sci., 2005, 11, 642-649. Structures of peptide agonists for human protease activated receptor 2. [136] Rink, R.; Arkema-Meter, A.; Baudoin, I.; Post, E.; Kuipers, A.; Nelemans, Bioorg. Med. Chem. Lett., 2012, 22, 916-919. S.A.; Akanbi, M.H.J.; Moll, G.N. To protect peptide pharmaceuticals against [150] Morrison, K.L.; Weiss, G.A. Combinatorial alanine-scanning. Curr. Opin. peptidases. J. Pharmacol. Toxicol. Methods, 2010, 61, 210-218. Chem. Biol., 2001, 5, 302-307. [137] Berezowska, I.; Chung, N.N.; Lemieux, C.; Wilkes, B.C.; Schiller, P.W. [151] Hruby, V.J. Designing peptide receptor agonists and antagonists. Nat. Rev. Dicarba analogues of the cyclic enkephalin peptides H-Tyr-c[D-Cys-Gly- Drug Discov., 2002, 1, 847-858. Phe-D(orL)-Cys]NH2 retain high opioid activity. J. Med. Chem., 2007, 50, [152] Savio, A.S.; Acosta, O.R.; Perez, H.G.; Alvarez, Y.R.; Chico, A.; Perez, 1414-1417. H.G.; Ojeda, M.O.; Aguero, C.A.; Estevez, M.; Nieto, G.G. Enhancement of [138] Stymiest, J.L.; Mitchell, B.F.; Wong, S.; Vederas, J.C. Synthesis of oxytocin the inhibitory effect of an IL-15 antagonist peptide by alanine scanning. J. analogues with replacement of sulfur by carbon gives potent antagonists with Pept. Sci., 2012, 18, 25-29. increased stability. J. Org. Chem., 2005, 70, 7799-7809. [153] U.S. National Institutes of Health. ClinicalTrials.gov. http://clinicaltrials.gov [139] Robinson, A.J.; Elaridi, J.; Van Lierop, B.J.; Mujcinovic, S.; Jackson, W.R. (Accessed June 25, 2012). Microwave-assisted RCM for the synthesis of carbocyclic peptides. J. Pept.

Received: March 29, 2012 Revised: July 04, 2012 Accepted: July 18, 2012